The present invention relates to the DNA sequences of the soybean peroxidase, and to the enzymatic assay of peroxidase activity. The invention further relates to the use of soybean peroxidase in immunoassays or oligonucleotide detection. The invention also relates to medical, environmental diagnostics and generally to oligonucleotides employing anti-soybean peroxidase monoclonal antibody. In addition, the present invention is directed to a promoter and regulatory sequences within the promoter. The present invention is also directed to DNA molecules including one or more of said regulatory sequences or full length promoter, such as a DNA construct comprising the regulatory region or full length promoter operably linked to one or more genes or antisense DNA. The invention is further directed to transformed plant tissue including the DNA molecule and to transformed plants and seeds thereof.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice of the invention, are incorporated by reference, and for convenience are respectively grouped in the appended list of references.
Function of Peroxidase in Biological Systems
Peroxidase is a class of proteins whose primary function is to oxidize a variety of hydrogen donors at the expense of peroxide or molecular oxygen. Areas where peroxidase could have an immediate use are: pulp and paper bleaching; on-site waste destruction; soil remediation; organic synthesis; and diagnostic chemistries.
At present, pulp and paper is bleached using chloride ions as a chemical agent. Soybean peroxidase has several advantages over chlorine bleach: lower cost; environmentally friendly; and hydroxyl ions produced by peroxidase have twice the oxidation power of chlorine ions.
In waste water and soil treatments, peroxidase has advantages since many organic compounds are toxic, inhibitory, or refractory to microbes, and certain organic compounds may result in the production of microbial products that produce toxic or offensive effluent.
The use of oxidation to achieve on-site destruction or detoxification of contaminated water and waste will increase in the future. If carried out to its ultimate stage, oxidation can completely oxidize organic compounds to carbon dioxide, water and salts.
Peroxidase has several uses in organic synthesis. Using peroxidase, researchers synthesized conductive polyaniline that produced only water as a by-product. Peroxidase can also be used in the manufacturing of adhesive and antioxidant intermediates.
Enzymes are now widely used in medical and environmental diagnostics. Horseradish peroxidase has been one of the most satisfactory enzymes but is relatively expensive. It has now been found that soybean peroxidase can be readily harvested from soybean hulls at minimal expense and be substituted for horseradish peroxidase in these diagnostic chemistries.
Several diagnostic chemistries using the enzymatic activity of horseradish peroxidase and polyclonal antibodies have been described in the literature. Horseradish peroxidase has been used for diagnostic determinations of various analytes and has been used as a label in enzyme labeled antibodies used in the determination of immunologically reactive species (i.e., immunoassays). Such determinations can be carried out in solution or in dry analytical elements.
One type of useful assay utilizes enzymatic reactions wherein the analyte, upon contact with the appropriate reagents, reacts with oxygen in the presence of a suitable enzyme to produce hydrogen peroxide in proportion to the concentration of the analyte. A detectable product such as a visible or fluorescent dye is then produced by the reaction of hydrogen peroxide in proportion to the concentration of the analyte in the tested liquids. Peroxidase is generally used in such assays to catalyze the oxidation of the interactive composition by hydrogen peroxide. One example of such an assay is a glucose assay using glucose oxidase. Glucose is oxidized in the presence of oxygen by the enzyme, glucose oxidase, to produce glucolactone and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide oxidizes a colorless dye such as tetramethylbenzidine to produce a colored product.
Another type of assay utilizes an immunologically reactive compound such as an antibody. These chemistries can be generally classified into two groups, namely, conjugate or enzyme labeled antibody procedures, and non-conjugate or unlabeled antibody procedures. In the conjugate procedures, the enzyme is covalently linked to the antibody and applied to a sample containing the immobilized antigen to be detected. Thereafter the enzyme substrate, e.g., hydrogen peroxide, and an oxidizable chromogen such as a leuco dye are applied. In the presence of the peroxidase, the peroxide reacts with the chromogen resulting in the production of color. The production of color indicates the presence and in some cases the amount of the antigen. In another method, a competing substance is used to dislodge an antibody enzyme conjugate from an immobilized substrate, leading to an absence of color.
In a method sometimes referred to as the sandwich assay or enzyme linked immunosorbent assay (ELISA), a first antibody is bound to a solid support surface and contacted with a fluid sample suspected to contain the antigen to be detected and an enzyme-antibody conjugate. The antigen complexes with the antibody and the conjugate bonds to the antigen. Subsequent introduction of the substrate and chromogen produces a visual indication of the presence of the antigen.
Procedures employing non-conjugated enzymes include the enzyme bridge method and the peroxidase-antiperoxidase method. These methods use an antiperoxidase antibody produced by injecting peroxidase into an animal such as a goat, rabbit or guinea pig. The method does not require chemical conjugation of the antibody to the enzyme but consists of binding the enzyme to the antigen through the antigen-antibody reaction of an immunoglobulin-enzyme bridge. In the enzyme bridge method a secondary antibody acts as an immunologic bridge between the primary antibody against the suspected antigen and the antiperoxidase antibody. The antiperoxidase antibody in turn binds the peroxidase which catalyzes the indicator reaction. In the peroxidase-antiperoxidase method, a complex of the peroxidase and the antiperoxidase antibody is formed. This complex can then be used in the immunologic bridge method.
Though peroxidase genes from different biologic sources have been identified, including other plant peroxidase genes from horseradish, tomato, pea, arabidopsis, peanut and turnip, and bacterial lignin peroxidase gene, there have not been any reports regarding identification of peroxidase genes from soybean.
Soybean coats are abundant and inexpensive, making them an excellent source of peroxidase. Therefore, there is substantial interest in cloning soybean peroxidase genes which will open the possibility of characterization of the expression patterns of individual peroxidase isoforms during normal plant development and genetic and molecular manipulations for increased peroxidase activity.
Regulation of Transcription and Translation
Eukaryotic genes consist of a transcription/translation initiation region, a coding region and a termination region. The transcription/translation initiation region is typically located upstream of the coding region, or in other words, entirely to the 5xe2x80x2 terminal end of the coding region. This initiation region includes a xe2x80x9cpromoterxe2x80x9d region, which contains the signals for RNA polymerase to begin transcription so that synthesis of the coded protein can proceed. In addition, there are xe2x80x9cuntranslated sequencesxe2x80x9d responsible for binding to ribosomes and translation initiation. The translation-related regions of these xe2x80x9cupstreamxe2x80x9d regulatory sequences vary in length and base composition from gene to gene and may be comprised of 100 bp or as much as 1 kbp.
The characteristics of the promoter will determine the level, tissue specificity and timing of transcription. Eukaryotic promoters are complex and are comprised of components which include a xe2x80x9cTATA boxxe2x80x9d at about 35 bp 5xe2x80x2 relative to the transcription start site. Further upstream, there can be a promoter element with homology to the consensus sequence CCAAT which, in plants, may be substituted by a consensus sequence which Messing et al. (1983) have termed the AGGA box. Additional DNA sequences in the 5xe2x80x2 untranslated region are believed to be involved in the modulation of gene expression. These include DNA sequences which control gene expression in a tissue-specific manner.
Through recombinant techniques, a plant transcription/translation initiation region can be designed to activate expression, by plant tissue, of a nucleic acid sequence of interest, such as a DNA sequence encoding a heterologous or non-naturally occurring gene. By modifying the promoter region of a construct capable of expression in a plant, the timing, tissue specificity and level of expression of transcription can be regulated.
The analysis of promoter-reporter gene fusions is one of the most widely used direct approaches to identify sequences that control the transcriptional regulation of plant genes. Regulatory elements that are involved in tissue-specific and/or developmentally regulated expression have been identified in many plant gene promoters (Mohan et al., 1993; Raghothama et al., 1993; lntapruk et al., 1994; Hatton et al., 1995; Sieburth and Meyerowitz, 1997). Gel retardation and DNA footprinting assays also have been used to study the transcriptional regulation of plant genes. Many nuclear proteins that bind to promoter fragments have been identified and genes encoding for these nuclear proteins have been isolated (Katagiri et al., 1989; Kawaoka et al., 1994; Zhao and Okita, 1995; Liu et al., 1998).
Peroxidase genes have been isolated from Arabidopsis thaliana (Intapruk et al., 1991), horseradish (Fujiyama et al., 1988 and 1990), tomato (Roberts and Kolattukudy, 1989), rice and wheat (Baga et al., 1995). Despite the role that plant peroxidases play in plant physiology, the regulatory mechanisms controlling peroxidase gene expression are not well understood. Little is known about the signaling factors or the DNA sequences that control peroxidase gene expression. Hormonal regulation of peroxidase gene expression has been reported in callus tissue, where the anionic peroxidases of potato and tomato were induced by abscisic acid at the transcriptional level (Roberts and Kolaftukudy, 1989). Lagrimini et al. (1991, 1996) demonstrated the importance of proper peroxidase regulation by over-expression and under-expression of an anionic peroxidase in tobacco, which in both cases resulted in aberrant phenotypes of the transgenic plants. Kawaoka et al. (1994) found that one trans-acting factor that interacts with a G-box element was essential for wound-induced expression of a horseradish peroxidase promoter. Intapruk et al. (1994) reported that multiple cis-elements in the horseradish peroxidase prxEa promoter were involved in regulating transcription of this peroxidase gene.
Recombinant Protein Technology
Recombinant protein technology has expanded to include protein production on a small scale for research purposes as well as large scale production processes for recombinant therapeutic proteins. The development of different protein expression systems reflects the variety of applications for their expressed products and the different features and functions of each. Many current production processes, such as cell culture and fermentation, are limited by poor yield, transient expression, poor folding and post-translational modification, costly manufacture, short shelf life and unpredictable immunogenic response.
Biotechnologists are starting to realize that higher organisms may be the most efficient hosts for production of recombinant proteins. The rapid progress made in development of cloning vectors for plants and animals has been allied with similar advances in cell culture systems for higher organisms. However, neither animal nor plant cells respond well to being suspended in culture media and therefore will attach to available solid support. The resulting cultures have much longer generation times than microbial cultures, limiting the yield of recombinant protein that can be obtained.
Another approach to production of recombinant protein is one that makes use of an intact organism rather than a cell culture. Examples of some methods known in the art for transformation of plant cells include: transformation via Agrobacterium tumefaciens, electroporation, microinjection and bombardment with DNA coated particles.
Within the plant biotechnology sector, there is great interest in expressing mammalian proteins in plants in a commercially feasible manner. One of the most important factors to be considered in developing a plant transformation procedure for production of recombinant proteins in plants is the availability of a promoter which provides expression in a tissue-specific manner. For example, for the transformation of plants with DNA encoding therapeutic proteins or vaccines, it is clearly desirable to obtain expression of the introduced gene in a tissue from which the protein product is readily recovered substantially free of other tissues.
The seeds of higher plants are very efficient at protein systhesis, as they accumulate large quantities of storage proteins and other compounds that the young seedling uses as a nutrient supply during the early stages of germination. Many crop plants have been bred specifically for the protein content of their seeds and the genes involved in seed development are quite well understood. If a gene for a natural seed protein is replaced by a gene coding for some useful foreign protein, the foreign protein may accumulate in the seeds. This has been demonstrated by the synthesis of pharmaceutical compounds called enkephalins in the seeds of engineered oilseed rape plants. Enkephalins are small proteins, only a few amino acids in length, and it has yet to be established that larger foreign proteins can be synthesized efficiently in the special environment found within the developing seed. Further success in this area would open up an exciting new area of biotechnology.